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A risk assessment study of plant genetic transformation usingAgrobacterium and implications for analysis of transgenic plants

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Abstract

Agrobacterium transformation systems forBrassica, Solanum andRubus, using carbenicillin, cefotaxime and ticaracillin respectively to eliminate contamination, were examined for the presence of residualAgrobacterium. The results indicated that none of the antibiotics in question, succeeded in eliminatingAgrobacterium and the contamination levels increased in explants from 12 to 16 weeks to such an extent thatSolanum cultures senesced and died. This may be due to the fact that four times the Minimum bactericidal concentration values (concentration to be used for elimination of contaminants in culture), for the three antibiotics, were higher than the concentrations employed in the culture medium. Contamination in shoot material decreased over 16 to 24 weeks possibly due to bacteriostatis and the use only of the apical node for further culture. The presence of the binary vector was also noted under non-selective conditions, even up to 6 months after transformation, where approx. 50% of contaminated material still harboured bacterial cells with the binary vector at levels of approx. 107 Colony forming units per gram.

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Abbreviations

CFU:

colony forming units

GUS:

β-glucoronidase

PCR:

polymerase chain reaction

MBC:

minimum bactericidal concentration

NPTII:

neomycin phosphotransferase

RAPD's:

random amplified polymorphic DNA

References

  • Armitage P, Walden R & Draper J (1988) Vectors for transformation of plant cells usingAgrobacterium. In: Draper J, Scott R, Armitage P & Walden R (eds) Plant Genetic Transformation and Gene Expression: A Laboratory Manual (pp 58–60). Blackwell Scientific Pubs., Oxford

    Google Scholar 

  • Barrett C & Cassells AC (1994) An evaluation of antibiotics for the elimination ofXanthomonas campestris pv.pelargonii (Brown) fromPelargonium xdomesticum cv. Grand Slam explantsin vivo. Plant Cell Tiss. Org. Cult. 36: 169–175

    Article  CAS  Google Scholar 

  • Barghchi M, Turgut K, Scott R & Draper J (1994) High frequency transformation from cultured cotyledons ofArabadopsis thaliana ecotypes “C24” and “Landsbergerecta”. Plant Growth Regulations 14: 61–67

    CAS  Google Scholar 

  • Bastaeiens L, Maene L, Harbaori Y, Van Sumere C, Van de Castelle KL & Debergh PC (1983) The influence of antibacterial products on plant tissue cultures. Med. Fac. Landbouwv. Rijsuniv. Gent. 48: 13–24

    Google Scholar 

  • Bertram J, Stratz M & Durre P (1991) Natural transfer of conjugative transposon Tn916 between gram - positive and gram - negative bacteria. J. Bacterial. 173(2): 443–448

    CAS  Google Scholar 

  • da Camara Machado ML, da Camara Machado A, Hanzer V, Weiss H, Regner F, Steinkellner H, Mattanovich D, Plail R, Knapp E, Kalthoff B & Katinger H (1992) Regeneration of transgenic plants ofPrunus armeniaca containing the coat protein gene of Plum Pox Virus. Plant Cell Rep. 11: 25–29

    Google Scholar 

  • Cassells AC, Harney MA, Carney BF, Mc Carthy E & Mc Hugh A (1988) Problems posed by cultivable bacteria and the endophytes in the establishment of axenic cultures ofPelargonium xdomesticum: The use ofXanthomonas pelargonii specific ELISA, DNA probes and culture indexing in the screening of antibiotic treated and untreated donor plants. Acta Hort. 225: 153–162

    Google Scholar 

  • Cassells AC (1991) Problems in tissue culture: culture contamination. In: Debergh PC & Zimmerman RH (eds) Micropropagation (pp 31–44). Kluwer Acad. Pubs, Netherlands

    Google Scholar 

  • Confalonieri M, Balestrazzi A & Bisoffi S (1994) Genetic transformation ofPopulus nigra byAgrobacterium tumefaciens. Plant Cell Rep. 13: 256–261

    Article  CAS  Google Scholar 

  • Dale PJ, Irwin JA & Scheffler JA (1993) Review: The experimental and commercial release of transgenic crop plants. Plant Breeding 111: 1–22

    CAS  Google Scholar 

  • De Bondt A, Eggermont K, Druart P, De Vil M, Goderis I, Vanderleyden J & Broekaert W (1994)Agrobacterium-mediated transformation of apple (Malus xdomestica Borkh.): An assessment of factors affecting gene transfer efficiency during early transformation steps. Plant Cell Rep. 13: 587–593

    Article  Google Scholar 

  • Draper J, Scott R & Hamil J (1988) Transformation of dicotyledonous plant cells using the Ti plasmid ofAgrobacterium nonefaciens and the Ri plasmid ofA. rhizogenes. In: Draper J, Scott R, Armitage P and Walden R (eds) Plant Genetic Transformation and Gene Expression: A Laboratory Manual (pp71–132). Blackwell Scientific Pubs, Oxford

    Google Scholar 

  • Eapen S & George L (1994)Agrobacterium tumefaciens mediated gene transfer in peanut (Arachis hypogaea L.). Plant Cell Rep. 13: 582–586

    Article  CAS  Google Scholar 

  • Genetello C, Van Larebeke N, Holsters M, De Picker A, Van Montagu M & Schell J (1977) Ti plasmids ofAgrobacterium as conjugative plasmids. Nature 260: 561–562

    Google Scholar 

  • Gonsalves C, Xue B, Yepes M, Fuchs M, Ling K, Namba S, Chee P, Slightim JL & Gonsalves D (1994) Transferring cucumber mozaic virus-white leaf strain coat protein gene intoCucumis melo L. and evaluating transgenic plants for protection against infections. J. Am. Soc. Hort. Sci. 119(2): 345–355

    CAS  Google Scholar 

  • Graham J & Mc Nicol R (1990) Plantlet regeneration and genetic transformation in soft fruit species. Acta Hort. 280: 517–522

    Google Scholar 

  • Grierson D & Covey S (1984) Genetic colonisation of plants byAgrobacterium. In: Plant Molecular Biology (pp 112–125). Blackie & Son Pubs., Glasgow

    Google Scholar 

  • Hamill J, Rounsley S, Spencer A, Todd G & Rhodes M (1990) The use of the polymerase chain reaction to detect specific sequences in transformed plant tissues. In: Nijkamp H J S, van der Plas L H W & van Aartijk (eds) Progress in Plant Cellular and Molecular Biology (pp 183–188). Kluwer Acadmic, Dordrecht.

    Google Scholar 

  • Hassan M, Swartz H, Inamine G & Mullineaux P (1993)Agrobacterium tumefaciens - mediated transformation of severalRubus genotypes and recovery of transformed plants. Plant Cell Tiss. Org. Cult. 33: 9–17

    Article  Google Scholar 

  • Hayward AC (1974) Latent infections by bacteria. Ann. Rev. Phytopath. 12: 87–97

    Article  Google Scholar 

  • Heinemann J & Sprague G (1989) Bacterial conjugative plasmids mobilise DNA transfer between bacteria and yeast. Nature 340: 205–209

    Article  CAS  PubMed  Google Scholar 

  • Higgins E, Hulme J & Shields R (1992) Early events in transformation of potato byAgrobacterium tumefaciens. Plant Science 82: 109–118

    Article  CAS  Google Scholar 

  • van der Hoeven C, Dietz A & Landsmann J (1994) Variability of organ-specific gene expression in transgenic tobacco plants. Trans. Res. 3: 159–165

    Google Scholar 

  • Horsch R & King K (1983) A covert contaminant of cultured plant cells: elimination of aHyphomicrobium sp. from culture ofDatura innoxia (Mill.). Pl. Cell Tiss. Org. Cult. 2: 21–28

    Google Scholar 

  • Howe GT, Goldfarb B & Strauss SH (1994)Agrobacterium - mediated transformation of hybrid poplar suspension cultures and regeneration of transformed plants. Plant Cell Tiss. Org. Cult. 36: 59–71

    Article  CAS  Google Scholar 

  • Hulme J, Higgins E & Shields R (1992) An efficient genotype - independent method for regeneration of potato plants from leaf tissue. Plant Cell Tiss. Org. Cult. 3: 161–167

    Google Scholar 

  • Jacq B, Lesobre O, Sangwan S & Sangwan - Noreel S (1993) Factors influencing T - DNA transfer inAgrobacterium - mediated transformation of sugarbeet. Plant Cell Rep. 12: 621–624

    Article  CAS  Google Scholar 

  • Jefferson R A (1987a) GUS gene fusion system user's manual. Plant Breeding Institute, Cambridge version 1.21

  • Jefferson R A (1987b) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol. Biol. Rep. 5(4): 387–405

    CAS  Google Scholar 

  • Jones JB & Raju BC (1988) Systemic movement ofAgrobacterium tumefaciens in symptomless stem tissue ofChrysanthemum morifolium. Plant Disease 72: 51–54

    Google Scholar 

  • Kerr A, Manigault P & Tempe J (1977) Transfer of virulence in vivo andin vitro inAgrobacterium. Nature 265: 560–561

    Article  CAS  PubMed  Google Scholar 

  • Leifert C, Camotta H, Wright S, Waites B, Cheyene V & Waites WM (1991) Elimination ofLactobacillus plantarum, Corynebacterium spp.,Staphlococcus saprophyticus andPseudomonas paucimobilis from micropropagatedHemercallis, Choisya andDelphinium cultures using antibiotics. J. App. Bact. 71: 307–330

    Google Scholar 

  • Lilley A, Fry J, Day J & Bailey M (1994)In situ transfer of an exogenously isolated plasmid betweenPseudomonas spp. in sugarbeet rhizosphere. Microbiology 140: 27–33

    CAS  Google Scholar 

  • Lorenz M & Wackernagel W (1991) High frequency of natural genetic transformation ofPseudomonas stutzeri in soil extract supplemented with a carbon/energy and phosphorus source. Applied and Environmental Microbiology 57(4:): 1246–1251

    CAS  PubMed  Google Scholar 

  • Maheswaran G, Welander M, Hutchinson JF, Graham MW & Richards D (1992) Transformation of apple rootstock M26 withAgrobacterium tumefaciens. J. Plant Physiol. 139: 560–568

    Google Scholar 

  • Martin GC, Miller AN, Castle LA, Moms JW & Dandekar AM (1990) Feasibility studies using the β-glucuronidase as a gene fusion marker in apple, peach and radish. J. Am. Soc. Hort. Sci. 115(4): 686–691

    CAS  Google Scholar 

  • Millam S (1989)Agrobacterium - mediated transformation systems of Brassica species. Aspects of Appl. Biol. 23: 23–30

    Google Scholar 

  • Mlynarova L, Bauer M, Nap J & Pretova A (1994) High efficiencyAgrobacterium - mediated gene transfer to flax. Plant Cell Rep. 13: 282–285

    CAS  Google Scholar 

  • Mogilner N, Zutra D, Gafny R & Bar - Joseph M (1993) The persistence of engineeredAgrobacterium tumefaciens in agroinfected plants. Molecular Plant - Microbe Interactions 6(50): 673–675

    CAS  PubMed  Google Scholar 

  • Moloney M, Walker J & Sharma K (1989) High efficiency transformation of Brassica napus usingAgrobacterium vectors. Plant Cell Rep. 8: 238–242

    Article  CAS  Google Scholar 

  • Murashige T & Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco. Physiol. Plantarum 15: 47–497

    Google Scholar 

  • Pugliesi C, Biasini MG, Fambrini M & Baroncelli S (1993) Genetic transformation byAgrobacterium in the interspecific hybridHelianthus annuus xHelianthus tuberosus. Plant Sci. 93: 105–115

    Article  CAS  Google Scholar 

  • Radke S, Andrews B, Moloney M, Crouch M, Kridl J & Knauf V (1988) Transformation ofBrassica napus L. usingAgrobacterium tumefaciens: developmentally regulated expression of a reintroduced napin gene. Theor. Appl. Gen. 75: 685–694

    Article  CAS  Google Scholar 

  • Sarria R, Calderon A, Thro AM, Tortes E, Mayer J & Roca WM (1994)Agrobacterium-mediated transformation ofStylosanthes guianensis and production of transgenic plants. Plant Sci. 96: 119–127

    Article  CAS  Google Scholar 

  • Schafer A, Kalinowski J, Simon R & Seep - Feldhaus A (1990) High - frequency conjugal plasmid transfer from gram - negativeEscherichia coli to various gram - positive Coryneform - bacteria. J. Bacteriol. 172(3): 1663–1666

    CAS  PubMed  Google Scholar 

  • Tarbah FA & Goodman RN (1987) Systemic spread ofAgrobacterium tumefaciens Biovar 3 in the vascular system of grapes. Phytopath. 77: 915–920

    Google Scholar 

  • Valles MP & Lasa JM Agrobacterium-mediated transformation of commercial melon (Cucumis melo L., cv. Amarillo Oro).Plant Cell Rep. 13: 145–148

    CAS  Google Scholar 

  • Wenzler H, Mignery G, May G & Park W (1989) A rapid and efficient transformation method for the production of large numbers of transgenic potato plants. Plant Sci. 63: 79–85

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. Teagasc, Kinsealy Research and Development Centre, Malahide Road, 17, Dublin, Ireland

    Carol Barrette

  2. Scottish Crop Research Institute, DD2 5DA, Invergowrie, Dundee, Scotland

    Emily Cobb, Ronald McNicol & Gary Lyon

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  1. Carol Barrette
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  2. Emily Cobb
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  3. Ronald McNicol
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  4. Gary Lyon
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Barrette, C., Cobb, E., McNicol, R. et al. A risk assessment study of plant genetic transformation usingAgrobacterium and implications for analysis of transgenic plants. Plant Cell Tiss Organ Cult 47, 135–144 (1997). https://doi.org/10.1007/BF02318949

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  • DOI: https://doi.org/10.1007/BF02318949

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